2-Keto-L-gulonic acid which is useful as an intermediate for synthesizing L-ascorbic acid has been produced by the industrially established so-called Reichstein method [see, Helvetica Chimica Acta, 17, 311 (1934)]. However, this method involves many steps and requires a large amount of solvent and, therefore, is insufficient for industrial technology of today.
On the other hand, instead of Reichstein method, several methods mainly employing microorganisms have been proposed. For example, a method which comprises subjecting D-glucose to microbiological oxidation to produce 5-keto-D-gluconic acid, reducing it chemically or microbiologically to obtain L-idonic acid and then oxidizing the resultant microbiologically to obtain 2-keto-L-gulonic acid [see, U.S. Pat. No. 2,421,611]; and a method which comprises oxidizing D-glucose microbiologically to obtain 2,5-diketo-D-gluconic acid, reducing it microbiologically or chemically to obtain 2-keto-L-gulonic acid [see, Japanese Patent Publication Nos. 39-14493, 53-25033, 56-15877 and 59-35920]have been investigated.
However, chemical reduction steps employed in these methods, i.e., the reduction of 5-keto-D-gluconic acid to idonic acid in the former method and the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid in the latter method are accompanied with problems in stereospecificity and they produce D-gluconic acid and 2-keto-D-gluconic acid as by-products, respectively, which results in decrease in yield. Further, when the above reduction is carried out microbiologically, excessive glucide should be supplied to the microorganisms as a reduction energy source, which also results in lowering of yield. In this respect, when L-sorbose is used as a starting material, 2-keto-L-gulonic acid can be produced only by an oxidation step.
In fact, several trials utilizing this advantage have been made by using bacteria belonging to the genera Gluconobacter, Pseudomonas, Serratia, Achromobacter and Alcaligenes [see, Biotechnology and Bioengineering, 14, 799 (1972); Japanese Patent Publication No. 41-159 and No. 41160; U.S. Pat. No. 3,043,749; USSR Patent No. 526,660; Japanese Patent Publication No. 49-39838; Acta Microbiological Sinica, 20, 246 (1980) and 21, 185 (1981); Japanese Patent Laid Open Publication No. 62-48389].
However, the disclosed strains give insufficient yield and therefore, they are insufficient for industrial use.
Recently, there has been reported a method for producing 2-keto-L-gulonic acid from D-glucose by using one bacterial strain obtained by introducing 2,5-diketo-D-gluconic acid reductase gene of a microorganism belonging to Corynebacterium into a microorganism belonging to Erwinia according to DNA recombination technique [see, Science, 230, 144 (1985)]. However, this method is also insufficient for utilizing industrial use from the viewpoint of the amount of 2-keto-L-gulonic acid produced.